29 TREATMENT OF DONOR CELLS WITH XENOPUS OOCYTE EXTRACT ENHANCED SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

Somatic cell nuclear transfer (SCNT) efficiency in pigs and other species is still very low. This low efficiency and the occurrence of developmental abnormalities in offspring has been attributed to incomplete or incorrect reprogramming. Cytoplasmic extracts from both mammalian and amphibian oocytes can alter the epigenetic state of mammalian somatic nuclei as well as gene expression to more resemble that of pluripotent cells. Rathbone et al. (2010) has showed that pretreating somatic donor cells with frog oocyte extract (FOE) increased live birth in ovine. Liu et al. (2011) also reported that treating donor cells with FOE enhanced handmade clone embryo development in pigs. The aim of this study was to evaluate the early development of cloned embryos produced with porcine GFP fibroblasts pre-treated with a permeabilizing agent, digitonin and matured frog oocyte extract. Frog egg cytoplasmic extract was prepared from one frog's oocytes after being matured in vitro to MII stage. The experiment included 2 groups. In the FOE-treated group, GFP-tagged fetal fibroblasts were permeabilized by digitonin (15 ng mL–1) and incubated in FOE containing an ATP-regenerating system (2.5 mM ATP, 125 μM GTP, 62.5 μg mL–1 of creatine kinase, 25 mM phosphocreatine and 1 mM NTP) at room temperature (24°C) for 2 h; cell membranes were re-sealed by culturing in 10% FBS in DMEM media for 2.5 h at 38.5°C before used as donor cells. In the control group, the same donor cells were treated with digitonin, but without frog oocyte extract incubation. The SCNT embryos were produced by using the 2 groups of donor cells as described above. In total, 305 control and 492 FOE oocytes were enucleated from 8 biological replicates. Two hundred fifty control and 370 FOE couplets were fused and cultured in porcine zygote medium 3. Percent cleavage was recorded on Day 2 and the percent blastocyst formation was determined on Day 7 (SCNT day = 0). In addition, the number of nuclei in the blastocysts was recorded on Day 7. Percent fusion, cleavage, blastocyst formation and number of nuclei in blastocysts were analysed by using SAS software (v9.2), with day and treatment class as main effects. There was no difference in percent fusion (FOE, 76.2 ± 2.5% vs control, 80.8 ± 2.8%) or in cleavage (FOE: 74.8 ± 2.5% vs control: 74.6 ± 2.9%). Only green blastocysts with 16 or more nuclei were considered to be a true SCNT blastocyst. The percent blastocyst was higher in the FOE group than that in the control (13.9 ± 0.8% vs 9.5 ± 0.9%, P < 0.05), whereas the number of nuclei in the blastocysts was not different between the 2 groups (39.7 ± 2.4, 35.9 ± 3.8 for FOE and control, respectively). In conclusion, our study demonstrated that pre-treatment of donor cells with digitonin and Xenopus MII oocyte extract increased porcine SCNT embryo development to blastocyst and cloning efficiency. Funded by the National Natural Science Foundation of China (NO. 31071311), Natural Science Foundation of Fujian Province of China (No. 2009J06017) and NIH U42 RR18877.

Frog oocyte in vitro maturation test as a method to investigate Ni2+ toxicity

Frog oocyte in vitro maturation test as a method to investigate Ni2+ toxicity The ability of gonadotropic hormone (GTH) of sturgeon fish Acipenser göldenstödti Br. to initiate a specific stimulatory effect on grass frog Rana temporaria L. oocytes, producing its in vitro maturation, was employed as a method to investigate the effect of nickel ion concentration on test oocytes in a highly sensitive test system, "Oocyte-GTH". Oocytes of four frogs with different hormone sensitivity were used in the investigation. It was shown that Ni2+ at a concentration of 0.1 mg/L produced a considerable stimulatory effect only in test frog oocytes with low hormonal sensitivity and maturation activity, while a Ni2+ concentration over 0.25 mg/L caused a significant decrease and produced even total blocking of the hormonal sensitivity and maturation ability of all oocytes, regardless of their initial hormonal sensitivity. The frog oocyte maturation test was shown to be a useful method to assess toxic effect of Ni2+ on water frog reproduction.

Novel Functions of Protein Arginine Methyltransferase 1 in Thyroid Hormone Receptor-Mediated Transcription and in the Regulation of Metamorphic Rate in Xenopus laevis

ABSTRACT Protein arginine methyltransferase 1 (PRMT1) acts as a transcription coactivator for nuclear receptors through histone H4 R3 methylation. The in vivo function of PRMT1 is largely unknown. Here we investigated the role of PRMT1 in thyroid hormone (T3) receptor (TR)-mediated transcription in vivo during vertebrate development. By using intestinal remodeling during T3-dependent Xenopus laevis metamorphosis for in vivo molecular analysis, we first showed that PRMT1 expression was upregulated during metamorphosis when both TR and T3 were present. We then demonstrated a role for PRMT1 in TR-mediated transcription by showing that PRMT1 enhanced transcriptional activation by liganded TR in the frog oocyte transcription system and was recruited to the T3 response element (TRE) of the target promoter in the oocyte, as well as to endogenous TREs during frog metamorphosis. Surprisingly, we found that PRMT1 was only transiently recruited to the TREs in the target during metamorphosis and observed no PRMT1 recruitment to TREs at the climax of intestinal remodeling when both PRMT1 and T3 were at peak levels. Mechanistically, we showed that overexpression of PRMT1 enhanced TR binding to TREs both in the frog oocyte model system and during metamorphosis. More importantly, transgenic overexpression of PRMT1 enhanced gene activation in vivo and accelerated both natural and T3-induced metamorphosis. These results thus indicate that PRMT1 functions transiently as a coactivator in TR-mediated transcription by enhancing TR-TRE binding and further suggest that PRMT1 has tissue-specific roles in regulating the rate of metamorphosis.

Promoter sequences required for transcription of Xenopus laevis histone genes in injected frog oocyte nuclei

Amphibian oogenesis is accompanied by the accumulation of histone mRNA and proteins in the absence of ongoing DNA replication. To begin an analysis of the mechanisms by which histone gene expression is regulated during frog oogenesis and embryogenesis, we used oocyte injection to examine the upstream sequences required for transcription of genes encoding each of the five histone classes. We found that sequences necessary for maximal levels of transcription are located 100 to 200 base pairs upstream of the corresponding start sites. In this region, each promoter examined contains conserved sequence elements, several of which seem to be histone gene class specific, in addition to other, more common sequence elements believed to be used by general transcription factors.